Diagnosis and treatment of bacterial dysentery

ABSTRACT

Diagnostic and therapeutic compositions which comprise the αGal(1-4)βGal subunit are described. These compositions permit the rapid diagnosis and treatment of enteric infections caused by E. coli that produce shiga-like toxins (SLT).

This application is a continuation of application Ser. No. 08/453,775,filed May 30, 1995, now U.S. Pat. No. 5,679,653, which in turn is adivisional of application Ser. No. 08/126,645, filed Sep. 27, 1993, nowU.S. Pat. No. 5,620,858, which further, in turn, is a continuation ofapplication Ser. No. 07/778,732, filed Oct. 18, 1991 and now abandoned.

TECHNICAL FIELD

The invention relates to diagnosis and treatment of diarrhea caused bybacterial infection. More specifically, the invention concerns detectionand neutralization of shiga-like toxins (SLT) associated with entericbacterial infection.

BACKGROUND ART

Diarrhea caused by strains of pathogenic E. coli has been found to beassociated with the production of a variety of enterotoxins. Somepathogenic E. coli enterohemorrhagic produce enterotoxins that areclosely related to the shiga toxin associated with Shigella-causeddysentery. The first member of the family of shiga-like toxins (SLT) tobe isolated was cytotoxic for African Green Monkey (Vero) cells and wasoriginally called verotoxin. Since its structural similarity to shigatoxin has been established by sequencing of the relevant genes, thistoxin is now more commonly called shiga-like toxin I (SLTI). See, forexample, Calderwood, S. B., et al., Proc Natl Acad Sci USA (1987)84:4364-4368; Jackson, M. P., et al., Micro Pathog (1987) 2:147-153;Strockbine, N. A., et al., J Bacteriol (1988) 170:1116-1122.

Additional members of the SLT family have subsequently been isolatedthat can be distinguished serologically, on the basis of gene sequenceor host specificity (Gannon, V. P. J., et al., J Gen Microbiol (1990)136:1125-1135; Weinstein, D. L., et al., J Bacteriol (1988)170:4223-4230; Ito, H., et al., Microb Pathog (1990) 8:47-60; Head, S.C., et al., FEMS Microbiol Lett (1988) 51:211-216; Schmitt, C. K., etal., Infect Immun (1991) 59:1065-1073; Scotland, S. M., et al., Lancet(1985) ii:885-886; Oku, Y., et al., Microb Pathog (1989) 6:113-122.Various types of SLTII have been described and have been assignedvarious designations depending on the strain of E. coli from which theyare isolated and the hosts they inhabit. Thus, variants have beendesignated SLTII; vtx2ha; SLTIIvh; vtx2hb; SLTIIc; SLTIIvp and so forth.

All of the SLT's are multimeric proteins composed of an enzymatic (A)subunit and multiple (B) subunits responsible for toxin binding toreceptors on host tissues. The binding B oligomers of SLTI, SLTII andSLTIIvh recognize host cell globoseries glycolipid receptors containingat minimum the disaccharide subunit αGal(1-4)βGal at the non-reducingterminus; SLTIIvp has been shown to bind to the receptors containingthis subunit but not necessarily at the non-reducing end (Samuel, J. E.,et al., Infect Immun (1990) 58:611-618; Boyd, B., et al., Nephron (1989)51:207-210; DeGrandis, S., et al., J Biol Chem (1989) 264:12520-12525;Waddell, T., et al., Biochem Biophys Res Comm (1988) 152:674-679;Lingwood, C. A., et al., J Biol Chem (1987) 262:8834-8839; Waddell, T.,et al., Proc Natl Acad Sci USA (1990) 87:7898-7901; Cohen, A., et al., JBiol Chem (1987) 262:17088-17091; Jacewicz, M., et al., J Exp Med (1986)163:1391-1404; Lindberg, A. A., et al., J Biol Chem (1987)262:1779-1785.

SLT activity has been detected in stool samples of symptomatic patients(Karmali, M. A., et al., J Clin Microbiol (1988) 22:614-619; Maniar, A.C., et al., J Clin Microbiol (1990) 28:134-135). However, there isdifficulty in detecting the presence of SLTs clinically since these arevery potent toxins present in low concentrations. In order to assuredetection, the SLT present in the sample must be concentrated to enhancethe reliability of the assay. Present diagnostic procedures aretechnically demanding, time consuming and of limited practical use inthe clinical setting. Thus there is a clear need for improved diagnosticclinically practical and rapid procedures.

Also, antibiotics are not recommended for treatment of enterohemorrhagicE. coli infection (Robson, W. L. M., et al., J Pediatr (1990)117:675-676) and the use of antimotility drugs also appears to becounterproductive (Cimolai, N., et al., J Pediatr (1990) 117:676. Thereis, therefore, also a clear need for new and effective therapeuticagents.

It has now been found that artificial substrates containing theαGal(1-4)βGal (P₁ disaccharide) subunit and more preferably theαGal(1-4)βGal(1-4)βGlcNAc (P₁ trisaccharide) or αGal(1-4)βGal(1-4)βGlc(P_(k) trisaccharide) subunit are effective in detecting andneutralizing members of the SLT family under conditions necessary toeffect recovery of the patient and as such represent novel therapeuticand diagnostic tools in the treatment of E. coli-mediated dysentery.

DISCLOSURE OF THE INVENTION

The invention provides compositions and methods that permit practicaland effective diagnosis of E. coli-caused enterotoxic bacterialinfections that present clinically as severe diarrhea, hemorrhagiccolitis, hemolytic uremic syndrome or thrombotic thrombocytopenicpurpura. The invention also provides compositions useful in the therapyof these and related conditions.

Thus, in one aspect, the invention is directed to a method to simply andrapidly bind to a support shiga-like toxins (SLT) from a biologicalsample at physiological conditions for diagnostic use, which methodcomprises contacting said sample with an effective amount of an affinitysupport wherein the affinity ligand comprises an αGal(1-4)βGal subunitunder conditions wherein said SLT is adsorbed to the affinity support;and detecting any SLT bound to the support.

In another aspect, the invention is directed to methods to detect thepresence of SLT in a biological sample which method comprises contactingsaid sample with a composition comprising the αGal(1-4)βGal subunitunder conditions wherein said subunit is complexed to any SLT present inthe sample and detecting the presence of any complex formed.

In a third aspect, the invention is directed to a method to treatenteric infections caused by microorganisms that produce one or moreSLTs, which method comprises administering to a subject in need of suchtreatment an effective amount of a composition comprising theαGal(1-4)βGal subunit.

In still other aspects, the invention is directed to pharmaceuticalcompositions which comprise the αGal(1-4)βGal subunit.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and B demonstrate the toxicity of bacterial extracts obtainedusing polymixin-B with respect to their ability to kill Vero cells inthe presence and absence of various SYNSORBs.

FIGS. 2A and B demonstrate the toxicity of bacterial extracts obtainedusing lysozyme with respect to their ability to kill Vero cells in thepresence and absence of various SYNSORBs.

FIG. 3A demonstrates that as little as 10 mg of P_(k) trisaccharideSYNSORB removes >90% SLT toxins from bacterial extracts.

B demonstrates that the binding of the SLT toxins occurred within 5minutes of mixing extracts with the P_(k) SYNSORB.

FIG. 4 demonstrates the difficulty in eluting the bound I¹²⁵ labelledSLTI from various SYNSORBs utilizing a variety of eluants.

FIG. 5 demonstrates that >90% SLTI, SLTII/IIc and SLTII activity wasneutralized by co-incubation of Vero cells and SLT extracts for threedays, with as little as 10 mg of P_(k) trisaccharide SYNSORB.

The subunit is bound preferably through a linking arm such as thatdescribed by Lemieux, R. U., et al., J Am Chem Soc. (1975) 97:4076-4083;to a solid, inert support that can be easily eliminated from thegastrointestinal system. An inert silica matrix embodiments of which arecommercially available as "SYNSORB™" are preferred.

MODES OF CARRYING OUT THE INVENTION

The compositions useful in the conduct of the methods of the inventioninclude a αGal(1-4)βGal disaccharide subunit, preferably theαGal(1-4)βGal (1-4)βGlcNAc trisaccharide subunit or αGal(1-4)βGal(1-4)βGlc trisaccharide subunit, preferably at the non-reducing terminusof an oligosaccharide. The di- and trisaccharides may be provided as aportion of a larger oligosaccharide coupled to a solid support orcoupled directly, preferably through a linking arm such as thatdescribed by Lemieux, R. U., et al., J Am Chem Soc (1975) 97:4076-4083.The di- and trisaccharide subunits may also be coupled directly topharmaceutically acceptable carriers or constitute a portion of anoligosaccharide coupled to such carriers. Depending on the applicationfor which the compositions of the invention are suggested, thecomposition is designed to accommodate the di- or trisaccharide subunitsso as advantageously to be employed in these applications.

As used herein, "shiga-like toxin" or "SLT" refers to group of toxinsproduced by enterohemorrhagic E. coli that resemble theShigella-produced shiga toxins as is commonly understood in the art.These toxins comprise an enzymatically active A subunit and a multimericreceptor binding B subunit. Such SLTs include SLTI and the variousgrouped toxins designated in the art SLTII.

Rapid, tight binding of SLT's to P₁ disaccharide, P₁ trisaccharide orP_(k) trisaccharide is demonstrated by the verocytoxicity neutralizationand I¹²⁵ binding assays contained herein. SYNSORB™ bearing haptens,e.g., the G_(M1) ganglioside Neu5Ac(2-3)βGal(1-4)βGlc and heat labiletoxin from enterotoxigenic E. coli, would be expected to behavesimilarly. A single SYNSORB™ bearing several haptens with specificityfor the different binding subunits of several different gastrointestinalinfections should now be possible. Such universal SYNSORB™s wouldprovide rapid, simple simultaneous diagnosis of a variety ofgastrointestinal disorders.

The SYNSORB™s employed were obtained from Chembiomed (Edmonton, Canada).In each case the 8-methoxycarbonyloctyl glycoside of the respectivehapten is activated and ligated to a silylaminated solid supportfollowed by acetylation of the remaining amine groups on the solidsupport. These formulations are sold commercially as "SYNSORB™"s:

"P₁ -di," which contains 0.60 μmole/g αGal(1-4)βGal disaccharide;

"P₁ -tri," which contains 0.91 μmole/g αGal(1-4)βGal(1-4)βGlcNActrisaccharide;

"P_(k) -tri," which contains 0.74 μmole/g αGal(1-4)βGal(1-4)βGlctrisaccharide;

"Linear B like tri," which contains 0.47 μmole/gαGal(1-3)βGal(1-4)βGlcNAc trisaccharide;

"Linear B like di," which contains 0.66 μmole/g αGal(1-3)βgaldisaccharide;

"Glucose mono," which contains 1.0 μmol B-glucose;8-methoxycarbonyoctanol activated and ligated to the silylated solidsupport.

A major aspect of the invention is the rapid efficient binding ofphysiological concentrations of any SLT present in biological samples,thus permitting assay of quantities of these toxins. Typically, in viewof the conditions with which these toxins are associated, the biologicalsample will be a stool sample. The sample is extracted and preparedusing standard extraction techniques and the extract is contacted with asolid support derivatized to an affinity ligand, wherein the affinityligand comprise the αGal(1-4)βGal disaccharide subunit, preferably theαGal(1-4)βGal(1-4)βGlcNAc trisaccharide subunit orαGal(1-4)βGal(1-4)βGlc trisaccharide subunit. Said contact may be in abatch treatment of the sample extract with the solid support, or thesolid support may be supplied as a chromatography column and the sampleextract applied to the column under conditions wherein any SLT presentin a sample is absorbed.

SLT may be measured directly on the surface of the SYNSORB™ using anysuitable detection system. In one approach, monoclonal or polyclonalantibodies specific for SLT can be utilized to quantify the amount ofSLT bound directly to SYNSORB™, labeled, for example, by radioactive,biotinylated, or fluorescent moieties. A wide variety of protocols fordetection of formation of specific binding complexes analogous tostandard immunoassay techniques is well known in the art.

Compositions containing the αGal(1-4)βGal disaccharide subunit,preferably the αGal(1-4)βGal (1-4)βGlcNAc trisaccharide subunit orαGal(1-4)βGal (1-4)βGlc trisaccharide subunit, that can also be used astherapeutic agents may be supplied wherein the disaccharide subunit ortrisaccharide subunits or an oligomer saccharide containing it iscoupled to a nontoxic carrier, such as a liposome, biocompatiblepolymer, or carrier analogous to the above-referenced SYNSORB™s.

Alternatively, the disaccharide or a larger moiety containing it as asubunit may be formulated in standard pharmaceutical compositions foradministration to the patient. Typically, the patient will be afflictedwith a diarrhetic condition, and the target SLT will be present in theintestinal tract. Thus, a suitable mode of administration is throughoral administration or other means of direct application to thegastrointestinal tract. The correct dosage range will depend on theseverity of the infection, the mode of administration, the mode offormulation, and the judgment of the attending practitioner.

Activity of the SLTs can be assayed by taking advantage of the toxicityof these compounds to Vero cells. Vero cells (ATCC CCL81) can beobtained from the American-Type Culture Collection, Rockville, Md. Theseare maintained at 37° C./5% CO₂ in minimal essential medium with Earl'ssalts (MEM, Gibco BRL, Gaithersburg, Md.) containing 3% fetal bovineserum (FBS). Confluent Vero cell monolayers are disrupted using 0.25%tissue-culture grade trypsin and approximately 10⁵ cells in 200 μlFBS-supplemented MEM are added to each well of a 96-well microtiterplate. The plates are incubated overnight at 37° C./5% CO₂.

The samples to be tested, and suitable controls are added to the variouswells and the plates are incubated for 2-3 days at 37° C./5% CO₂.Cytotoxic effects are readily visible in successful candidate wells ascompared to control wells. The results can be quantitated by aspiratingthe remaining liquid from each of the wells and fixing the Vero cellswhich remain viable with 95% methanol and staining with Geimsa stain.The results are recorded using a microtiter plate reader set at awavelength of 620 nm, as described by Samuel, J. E., Infect Immun (1990)58:611-618 (supra). The dilution of the candidate test solution. Thedilution of samples resulting in 50% destruction (CD₅₀) of themonolayers is determined by extrapolation from the resulting Vero cellkilling curves.

The following examples are intended to illustrate but not to limit theinvention.

EXAMPLE 1 SYNSORB--Verocytotoxicity Neutralization Assays

E. coli strains 0157:H-(E32511), which produces SLTII/SLTIIc and 026:H11(H19) which produces SLTI only, or strain C600(933W), which producesSLTII only, were grown overnight at 37° C. on tryptic soy broth (Difco,Detroit, Mich.) agar plates. Polymycin and lysozyme extracts wereprepared as described previously Karmali, M. A., et al., J ClinMicrobiol (1985) 22:614-619 and Head, S., et al., Infect Immunol (1990)58:1532-1537)!.

The first neutralization assay was designed to test the ability ofSYNSORBs to absorb SLT activity from the E. coli extracts. The assayinvolved incubating 1 mL of the E. coli extracts for 30 min. at roomtemperature in 1.5 mL microcentrifuge tubes (Fisher) with 2 to 50 mgSYNSORB on an end-over-end rotator. The tubes were then removed from theapparatus and after the SYNSORB had settled to the bottom (a fewseconds), serial five-fold dilutions of the absorbed extracts wereprepared in unsupplemented MEM. Twenty (20) μL of each dilution wasadded to the appropriate wells in 96 well microtiter plates containingVero cells. Bacterial extracts to which no SYNSORB was added served ascontrols. Once cytotoxic effects became apparent (2 to 3 days in theincubator) the growth medium was aspirated from each of the wells andVero cells which remained viable were fixed with 95% methanol andstained with Giensa stain (Fisher). The results were then recorded usinga microtiter plate reader set at a wavelength of 620 nm as describedpreviously Samuel et al., Infect Immun. 58:611-618 (1990)!. Theabsorbance data were then plotted versus the logarithm of the extractdilution. The dilution of the extracts resulting in 50% destruction(CD₅₀) of the monolayers was determined by extrapolation from theresulting Vero cell killing curves. Individual experiments were alwaysperformed in duplicate and, unless otherwise indicated, repeated atleast two times. The percentage of neutralization was computed from theequation: 100-(100 CD₅₀ oligosaccharide SYNSORB-treated extracted+CD₅₀acetylated silyl-aminated (ASA) SYNSORB-treated extract!). Thenon-parametric Mann-Whitney test using the two-tailed statistic T wasemployed to compute the significance level of difference between groupsof independent observations Altman, D. G., Practical statistics formedical research, 1st ed. New York, Chapman and Hall: 179-228 (1991)!.

The second neutralization assay (co-incubation assay) was designed totest the ability of Pk trisaccharide SYNSORB to protect Vero cells fromSLT activity over 3 days at 37° C. This assay involved incubating 180 μLof serial five-fold dilutions of polymyxin extracts in ethyleneoxide-sterilized 1.5 mL microcentrifuge tubes each containing 2, 5 or 10mg of Pk trisaccharide SYNSORB. After 1 h incubation with SYNSORB, theentire contents of each microcentrifuge tube were added to Vero cellsmonolayers in microtiter plates prepared as described above. Themicrotiter plates prepared as described above. The microtiter plateswere then incubated at 37° C. for 3 days and the results of theexperiment were recorded as described above (FIGS. 1 and 2).

The foregoing determination was repeated using varying amounts of Pk-triand various times of incubation, with the results shown in FIGS. 3A and3B. As shown in FIG. 3A, as little as 5 mg SYNSORB was capable ofneutralizing the activity of the extracts of both E32511 and H19strains; similarly, as shown in FIG. 3B, only about 5 min incubation wasrequired to achieve this result in either extract.

EXAMPLE 2 Iodinated SLT I Binding Assay

Purified SLTI was iodinated in 12×75 mm acid-washed glass culture tubescoated with 40 μg of Iodo Gen (Pierce Chemical Co., Rockford, Ill.).About 6 μg of purified SLTI was incubated for 1 min with 20 MBq ¹²⁵ -Ilabeled sodium iodide in 100 μl PBS. The reaction mixture was passedthrough a glass wool-plugged Pasteur pipette into 200 μl PBS containinga solution of cysteine (1 mg/ml) in PBS as described by Armstrong, G.D., et al., Infect Immunol (1987) 55:1294-1299. After 1 min, 200 μl ofPBS containing 1% BSA was added to the mixture and the iodinated SLTIwas purified by passing the solution through a 1 cm×30 cm Sephadex-G 25gel filtration column with 0.1% BSA in PBS. The efficiency of theiodination reaction was determined by measuring the number of countsthat were incorporated into trichloroacetic acid precipitated protein.Aliquots of the iodinated SLTI were stored at -90° C.

The assays were performed in PBS containing 0.1% BSA to reducenonspecific binding. 2 mg of the various SYNSORBs were incubated for 30on an end-over-end rotator with approximately 20,000 dpm of theiodinated SLTI prepared in Preparation B above (specific activity,2.2×10⁷ dpm/μg, CD₅₀ in the Verocytotoxicity assay, 0.4 pg/ml), in 0.5ml PBS/BSA). The SYNSORBs were then washed with 3×1 ml portions of PBSBSA to remove unbound counts. The derivatized SYNSORBs were counted inan LKB Rackgamma model 1270 Gamma Counter.

The results are shown in Table 1.

                  TABLE 1    ______________________________________    SYNSORB       % SLTI Bound    ______________________________________    Pk-tri        93    # 115         21    Glc            9    ASA            5    ______________________________________

The SLT bound to Pk-tri SYNSORB could be partially released using 0.1 Macetic acid, 6 M guanidine HCl, or by heating in boiling water bath for30 min in 10% SDS. However, neither 0.5 M lactose, 0.5 M galactose, or0.2 M EDTA could displace the bound SLTI (FIG. 4).

Subsequent experiments showed that 2 mg of Pk-tri neutralizedapproximately 90% of the activity in E. coli H19 (SLTI) but about 10 mgPk-tri SYNSORB was required to neutralize the activity of the E. coli32511 (SLTII/SLTIIc) or E. coli C600/933W (SLTII) to a similar extent(FIG. 5).

We claim:
 1. A pharmaceutical composition useful in treating entericinfections mediated by SLT which composition comprises as activeingredient a moiety comprising the disaccharide subunit αGal(1-4)βGal,which moiety is covalently attached to a solid inorganic support andwhich moiety is capable of binding SLT when so attached to said support,in admixture with a pharmaceutically acceptable excipient.
 2. Thepharmaceutical composition of claim 1 wherein said moiety comprises thetrisaccharide subunit αGal(1-4)βGal(1-4)βGlcNAc or the trisaccharidesubunit αGal(1-4)βGal(1-4)βGlc.
 3. The pharmaceutical composition ofclaim 1 wherein said support is an inert silica matrix.
 4. Thepharmaceutical composition of claim 1 which is capable of beingeliminated from the gastrointestinal tract.
 5. The pharmaceuticalcomposition of claim 1 wherein said moiety is attached to said supportvia a linker.
 6. The pharmaceutical composition of claim 5 wherein saidlinker comprises a --(CH₂)₈ C(O)-- linking arm.
 7. A pharmaceuticalcomposition suitable for oral administration to a subject and whichcomposition is useful in treating enteric infections medicated by SLTwhich composition comprises as active ingredient a compositioncomprising a pharmaceutically acceptable orally deliverable solid inertaffinity support capable of being eliminated from the gastrointestinaltract which support has an affinity ligand covalently attached theretothrough a spacer arm, which spacer arm is of corresponding length to--(CH₂)₈ C(O)-- and has a functional group at one terminus to react withsaid affinity ligand and a functional group at the other terminus toreact with said support, wherein said ligand is an oligosaccharidecomprising the disaccharide subunit αGal(1-4)βGal which binds the SLT inadmixture with a pharmaceutically acceptable excipient.
 8. Thepharmaceutical composition of claim 7 wherein said oligosaccharidecomprises the trisaccharide subunit αGal(1-4)βGal(1-4)βGlcNAc or thetrisaccharide subunit αGal(1-4)βGal(1-4)βGlc.
 9. The composition ofclaim 7 wherein said spacer arm corresponds to --(Ch₂)₈ C(O)--.
 10. Apharmaceutical composition useful in treating enteric infectionsmediated by SLT which composition comprises the trisaccharide subunitαGal(1-4)βGal(1-4)βGlc, wherein said subunit is covalently attached toan inert silica matrix via a --(CH₂)₈ C(O)-- linking arm, in admixturewith a pharmaceutically acceptable excipient.